Use of microvesicles in the treatment of medical conditions

ABSTRACT

This invention relates generally to populations of microvesicles containing or otherwise associated with viral particles, methods of producing these purified populations, and methods of using these purified populations in a variety of diagnostic, therapeutic and/or prophylactic indications.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/858,629 filed Apr. 8, 2013, pending, which is a ContinuationApplication of U.S. patent application Ser. No. 13/580,509, filed Aug.22, 2012, abandoned, which is a National Stage Entry of InternationalApplication No. PCT/US2011/025861, filed Feb. 23, 2011, which claims thebenefit of U.S. Provisional Application No. 61/307,213, filed Feb. 23,2010, the contents of each of which are hereby incorporated by referencein their entirety.

FIELD OF THE INVENTION

The invention provides purified populations of microvesicles containingor otherwise associated with viral particles, methods of producing thesepurified populations, and methods of using these purified populations ina variety of diagnostic, therapeutic and/or prophylactic indications.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 19, 2019, isnamed 030258-067963USD1_SL.txt, and is 1,350 bytes in size.

BACKGROUND OF THE INVENTION

Microvesicles are shed by eukaryotic cells, or budded off of the plasmamembrane, to the exterior of the cell. These membrane vesicles areheterogeneous in size with diameters ranging from about 10 nm to about5000 nm. The small microvesicles (approximately 10 to 1000 nm, and moreoften 30 to 200 nm in diameter) that are released by exocytosis ofintracellular multivesicular bodies are sometimes referred to in the artas “microvesicles.” Microvesicles contain nucleic acids that have beenused as biomarkers for medical diagnosis, prognosis and therapyevaluation.

There exists a need to further investigate the characteristics ofmicrovesicles and to exploit the capabilities of these microvesicles ina variety of therapeutic and prophylactic indications.

SUMMARY OF THE INVENTION

The purified populations, compositions and methods of the invention usenaturally secreted microvesicles to encapsulate, coat or otherwiseassociate with a viral vector particle to produce amicrovesicle-associated vector, referred to herein as “MAV”. Forexample, the viral particle can be encapsulated within a microvesicle,the viral particle can be associated with the microvesicle lipidmembrane, the viral vector can be associated with one or more types ofmembrane-bound proteins or receptors on the microvesicle surface and/orinterior, or any combination of these associations can be used toproduce a MAV. The association between the microvesicle and the viralparticle can be a covalent interaction or a non-covalent interaction.

The association between the microvesicle and the viral particle protectsor otherwise shields the viral particle. For example, the microvesicleshields the viral particle from an anti-virus immune response in vivo.The association between the microvesicle and the viral particle preventsor otherwise impedes the ability of neutralizing antibodies and/orcomplement components in a subject to bind or otherwise interact withthe viral particle. Depending on the microvesicle type, the microvesiclecomponents, including, for example, cytokines, cell surface receptors,may abrogate cytotoxic immunity against vector transduced cells types.

The purified populations of microvesicles associated with a viralparticle are useful to deliver a nucleic acid to a cell. In someembodiments, the cell is within the body of an individual. The cell towhich the nucleic acid gets delivered is referred to as the target cell.The viral particle can be engineered to contain a nucleic acid that itwould not naturally contain (i.e. which is exogenous to the normalcontent of the viral particle). In some embodiments, the cell whichproduces the microvesicle for administration is of the same or similarorigin or location in the body as the target cell. For example, fordelivery of a microvesicle to a brain cell, the cell which produces themicrovesicle would be a brain cell (e.g. a primary cell grown inculture). In other embodiments, the cell which produces the microvesicleis of a different cell type than the target cell. In one embodiment, thecell which produces the microvesicle is a type that is locatedproximally in the body to the target cell.

The nucleic acid sequence which can be delivered to a cell via amicrovesicle and associated viral vector can be RNA or DNA, and can besingle or double stranded, and can be selected from a group comprising:nucleic acid encoding a protein of interest, oligonucleotides, nucleicacid analogues, for example peptide-nucleic acid (PNA),pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc. Suchnucleic acid sequences include, for example, but are not limited to,nucleic acid sequences encoding proteins, for example that act astranscriptional repressors, antisense molecules, ribozymes, smallinhibitory nucleic acid sequences, for example but are not limited toRNAi, shRNA, siRNA, miRNA, antisense oligonucleotides, and combinationsthereof. The nucleic acid sequence can include or be accompanied byaccessory nucleic acid sequences, i.e., sequences needed for theexpression of the nucleic acid sequence. These accessory nucleic acidsequences include, for example, promoters, positive regulatory elementsand negative regulatory elements.

The microvesicles are shed from cells referred to herein as “producercells.” The producer cells can naturally shed one or more types ofmicrovesicles. Alternatively or in addition, the producer cells can bemodified, e.g., genetically engineered or otherwise modified usingcompounds that can affect microvesicle secretion and/or production, toshed one or more desired types of microvesicles.

Suitable producer cells for use in the purified microvesiclepopulations, compositions and methods of the invention include, by wayof non-limiting example, cells such as 293T (ATCC, see alsoBiotechniques. 2003 January; 34(1):184-9); Per.C6 (Crucell), AGE1.CR(ProBioGen AG); AGE1.HNO cell line (ProBioGen AG); and KG-1 cells (ATCC,see also Biochem Biophys Res Commun. 2005 Aug. 5; 333(3):896-907), whichcan be differentiated into human dendritic cells. Suitable producercells also include tumor cells such as cells from ovarian cancer,glioma, hepatocarcinoma (see e.g., PLoS One. 2010 Jul. 22; 5(7):e11469),as tumor-derived microvesicles have been shown to have immunosuppressiveproperties and can be useful for long-term expression of a transgene.Suitable producer cells also include regulatory T cell lines such asCD4+ CD25+ T cells (see e.g., Blood. 2005 Nov. 1; 106(9):306873. Epub2005 Jul. 14), which can be useful for long-term expression of atransgene.

In some embodiments, the lipid membrane of the microvesicle is modifiedto enhance or otherwise alter a property of the microvesicle, such as,for example, target cell type, cell activation, or a transductionproperty. For example, the expression or presence of a cell surfaceprotein found on the microvesicle can be altered to induce a change inthe microvesicle. In some embodiments, the surface of the microvesiclecan be modified to include a receptor ligand that targets a desired celltype or a bridging molecule linked to a receptor ligand that targets adesired cell type.

In some embodiments, the lipid membrane of the microvesicle can bemodified to express or include Biotin acceptor peptide-transmembrane(BAP-TM) on its lipid membrane (see e.g., Tannous et al. Nat Methods.2006 May; 3(5):391-6). The use of BAP-TM allows for display of anybiotinylated ligand or other protein on the membrane surface via astreptavidin bridge, as BAP is genetically fused with PDGR transmembranedomain and gets biotinylated using BirA expression.

In some embodiments, the lipid membrane of the microvesicle can bemodified to express or include streptavidin-conjugated magneticnanoparticles (see e.g., J Neurosci Methods. 2011 Jan. 15; vol.194(2):316-20). The use of streptavidin-conjugated magneticnanoparticles links magnetic particles to BAP-TM and then directs themicrovesicle to wherever a strong magnet is placed.

In some embodiments, the lipid membrane of the microvesicle can bemodified to express or include the membrane proteins from envelopedviruses. For example, vesicular stomatitis virus glycoprotein G (VSV-G)(see e.g., Proc. Natl. Acad. Sci. USA Vol. 93, pp. 15266-15271, December1996) can be used to pseudotype lentivirus and retrovirus vectors, andVSV-G gives broad cell tropism. The use of VSV-G allows for robusttransgene expression in a variety of cultured cells as well as geneexpression in vivo.

In some embodiments, the lipid membrane of the microvesicle can bemodified to express or include transferrin mimic peptide (CRTIGPSVC (SEQID NO: 4)) (see e.g., J Clin Invest. 2011 Jan. 4; 121(1):161-73).CRTIGPSVC (SEQ ID NO: 4) is useful in treating disorders of the brain,such as, for example, brain tumors, because CRTIGPSVC (SEQ ID NO: 4) isable to cross the blood-brain barrier. CRTIGPSVC (SEQ ID NO: 4) binds totransferrin receptor expressed on brain and brain tumor vasculature.Biotinylated CRTIGPSVC (SEQ ID NO: 4) can be linked to BAP-TM via astreptavidin bridge.

In some embodiments, the lipid membrane of the microvesicle can bemodified to express or include a transmembrane bound single chainantibody against EGFRvIII, which is overexpressed on many human gliomatumors. EGFRvIII can be used as a target for gene therapy-mediatedkilling of glioma cells.

In some embodiments, the lipid membrane of the microvesicle can bemodified to express or include CD40 ligand (CD4OL), which is specificfor CD40 expressed on the surface of dendritic cells and B cells. Theuse of the CD40/CD4OL interaction may enhance antigen presentation ofvectored antigens.

In some embodiments, the lipid membrane of the microvesicle can bemodified to express or include Rabies derived peptide (see e.g., Nature.2007 Jul. 5; 448(7149):39-43. Epub 2007 Jun. 17). Rabies derived peptidebinds to acetylcholine receptor on neurons and can be useful fortargeting therapeutic molecules to the CNS.

The microvesicles in the purified populations provided herein contain orare otherwise associated with the viral particles to produce a MAV. Thecharacteristic of a MAV according to the invention is a viral particle,e.g., viral capsid, that contains a nucleic acid encoding a gene ofinterest, where the MAV is further surrounded by or otherwise associatedwith a membrane derived from a virus-producing cell. For example, theMAV is or is derived from adenovirus, including replication defectiveand competent vectors; lentivirus; retrovirus; herpes virus, includingreplication defective and competent vectors; adeno-associated virus(AAV); alphavirus, including, for example, sindbis virus, semliki forestvirus, venezuelan equine encephalitis virus, Ross River virus;flavivirus vectors; baculovirus; bacteriophage; orthomyxovirus(influenza); vaccinia; human papilloma virus, including, for example,virus like particles; and paramyxovirus, including, for example,Newcastle disease virus vectors.

These viral particles are useful in a variety of applications such as,for example, gene transfer applications, including gene replacement,gene repair, and/or mRNA knockdown therapeutic applications, and vaccineapplications. These MAV are useful in gene transfer applications whereconventional viral vectors including, for example, conventional AAVvectors, are inefficient or incapable of infection of a target cell ortissue type.

In some embodiments, the vector in the MAV is an adeno-associated viral(AAV) vector. The AAV capsids are incorporated into microvesicles thatcan be purified from the supernatant of the producer cell line. Thesemicrovesicles are fully competent for transduction of cells in cultureas well as in vivo. In vivo delivery of the vector is markedly enhancedwith the associated microvesicles and extended to multiple tissuescompared to AAV capsids of those same serotypes as assessed by in vivobioluminescence imaging.

The purified populations of microvesicles and compositions containingthese purified populations are useful in a variety of therapeuticindications. The ability of vector producer cells to insert viralparticles inside microvesicles or the vector's association withmicrovesicles components (on the surface and/or interior) offer manyopportunities for gene therapy applications. First, the viral vector maybe co-delivered with therapeutic proteins, mRNA or microRNA inside themicrovesicle. The microvesicles are useful for enhancing the potency andbioavailability of a poorly soluble anti-inflammatory drug (see e.g.,Mol Ther. 2010 September; 18(9):1606-14. Epub 2010 Jun. 22). Second, asother viral particles, e.g. HIV, can be packaged in or otherwiseassociated with microvesicles (see e.g., PNAS, Jan. 17, 2006, vol. 103,no. 3: 738-743), the purified populations, compositions and methods ofthe invention use other gene therapy vectors, e.g. adenovirus,lentivirus, to obtain novel microvesicle gene delivery vehicles.Packaging within or other association with microvesicle membranes hasseveral advantages, as it allows targeting molecules incorporated intothe plasma membrane to be used for targeting of the microvesicles, andfurthermore, the microvesicles may shield the particle from pre-existingneutralizing antibodies or T cells in vivo. Microvesicles have beenobserved to have both immunogenic and immunosuppressive activitiesdepending on the donor cell type, they can be used to obtain higherimmune responses in vaccination strategies, or in cases where immuneresponses to the virus proteins may be problematic, microvesicles withimmunosuppressive properties can be used.

In some embodiments, the purified populations and compositions providedherein are useful in the treatment of a cancer or other neoplasticcondition, such as, for example, gliomas and other cancers of thecentral nervous system.

In some embodiments, the purified populations of microvesiclesassociated with a packaged or otherwise associated viral vector is aningredient (e.g., population of MAV as the active ingredient) in apharmaceutically acceptable composition and/or formulation suitable foradministration to the subject. Generally these compositions andformulations comprise a pharmaceutically acceptable carrier for theactive ingredient. The specific carrier will depend upon a number offactors, including for example, the route of administration.

In some embodiments, the invention includes a purified population ofmicrovesicles that contain or are otherwise associated with one or moreviral particles, wherein the microvesicle is shed or otherwise producedby a producing cell. In some embodiments, the microvesicles areassociated with the viral particles through a covalent interaction. Insome embodiments, the microvesicles are associated with the viralparticles through a non-covalent interaction. In some embodiments, theproducer cell naturally sheds the microvesicles. In some embodiments,the producer cell has been modified to shed the microvesicles. In someembodiments, the viral particle is or is derived from adenovirus,lentivirus, herpes virus, and adeno-associated virus (AAV). In someembodiments, the microvesicle comprises a lipid membrane having an outersurface that has been modified to include or express a receptor ligandor bridging molecule linked to a receptor ligand that targets a desiredcell type. In some embodiments, the desired target cell type isdifferent that the cell type that is target by a microvesicle having anunmodified lipid membrane. In some embodiments, the population ofmicrovesicles comprises about 10⁹ to 10¹³ genome copies. In someembodiments, the viral particle comprises a nucleic acid encoding apeptide, polypeptide or protein. In some embodiments, the viral particlecomprises a non-native nucleic acid.

In some embodiments, the invention provides methods of producing apurified population of microvesicles that contain viral particles byengineering a cell that sheds microvesicles, wherein the cell isengineered to comprise a viral vector and a nucleic acid encoding adesired polypeptide under the control of the viral nucleic acidsnecessary for expression of the desired polypeptide. The nucleic acidsequence can include or be accompanied by accessory nucleic acidsequences, i.e., sequences needed for the expression of the nucleic acidsequence. These accessory nucleic acid sequences include, for example,promoters, positive regulatory elements and negative regulatoryelements. In some embodiments, the method also includes the step ofincreasing production of microvesicles by exposing the engineered cellto a stimulus or by genetically engineering the engineered cell toincrease production of the microvesicles. In some embodiments, thestimulus is a chemical stressor or an environmental stressor. In someembodiments, the method also includes the step of increasingtransfection efficiency of the viral particles. In some embodiments, theengineered cell is further modified to express a targeting protein on anouter surface of the microvesicle.

In some embodiments, the invention provides uses of the purifiedpopulations of microvesicles that are associated with viral particles inthe treatment of a disorder in a subject. In some embodiments, thesubject is human. In some embodiments, the disorder is a cancer. In someembodiments, the cancer is a cancer of the central nervous system (CNS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting how AAV purified by standard techniques doesnot pellet efficiently using the centrifugation speed used formicrovesicle pelleting.

FIG. 2 is a graph depicting the ability of AAV associated-microvesiclesto efficiently transduce cells in culture.

FIGS. 3A and 3B are an illustration and a graph depicting the ability ofAAV associated-microvesicles to efficiently transduce cells in vivo.

FIGS. 4A-4E are a series of illustrations depicting the use of anti-AAVimmunogold labeling in the detection of microvesicle-associated vectors.In these illustrations, the arrows are indicating the location of AAV(AAV1 or AAV2) that is “free,” i.e., outside of a microvesicle, thetriangles are indicating the location of AAV (AAV1 or AAV2) inside amicrovesicle, and the diamond-headed arrows are indicating the locationof AAV (AAV1 or AAV2) that is bound to surface of a microvesicle. FIGS.4A and 4B depict the isolation and identification of AAV1 MAV, whileFIGS. 4C-4E depict the isolation and identification of AAV2 MAV.

DETAILED DESCRIPTION

The purified populations, compositions and methods described hereinprovide efficient and effective gene delivery systems based on viralvectors and microvesicles.

Microvesicles are shed by eukaryotic cells, or budded off of the plasmamembrane, to the exterior of the cell. These membrane vesicles areheterogeneous in size with diameters ranging from about 10 nm to about5000 nm. The small microvesicles (approximately 10 to 1000 nm, and moreoften 30 to 200 nm in diameter) that are released by exocytosis ofintracellular multivesicular bodies are sometimes referred to in the artas “microvesicles.” The methods and compositions described herein areequally applicable to microvesicles of all sizes; preferably 30 to 800nm; and more preferably 30 to 200 nm. In some of the literature, theterm “microvesicle” also refers to protein complexes containingexoribonucleases which are involved in mRNA degradation and theprocessing of small nucleolar RNAs (snoRNAs), small nuclear RNAs(snRNAs) and ribosomal RNAs (rRNA) (see e.g., Liu et al.,“Reconstitution, activities, and structure of the eukaryotic RNAmicrovesicle,” Cell, vol. 127:1223-37 (2006); van Dijk et al., “Humancell growth requires a functional cytoplasmic microvesicle, which isinvolved in various mRNA decay pathways,” RNA, vol. 13:1027-35 (2007)).Such protein complexes do not have membranes and are not “microvesicles”or “microvesicles” as those terms are used herein.

Microvesicles are shed from both normal and malignant cells.Microvesicles have been found in bodily fluids such as blood, milk,ascites, urine and saliva among other fluids. The interior content ofthe microvesicles vary between cell types. The membrane surrounding themicrovesicles, also referred to herein as the exterior content of themicrovesicles, varies between cell types. The exterior content of themicrovesicle can be modulated or otherwise manipulated to achieve adesired biological activity, such as, for example, targeting themicrovesicle to a specific cell type.

Small microvesicles shed by cells are known as “microvesicles” (Thery etal., 2002). Microvesicles are reported as having a diameter ofapproximately 30-100 nm and are shed from many different cell typesunder both normal and pathological conditions (Thery et al.,“Microvesicles: composition, biogenesis and function,” Nat Rev Immunol.,vol. 2:569-79 (2002)). Microvesicles are classically formed from theinward invagination and pinching off of the late endosomal membrane.This results in the formation of a multivesicular body (MVB) laden withsmall lipid bilayer vesicles (−40-100 nm in diameter), each of whichcontains a sample of the parent cell's cytoplasm (Stoorvogel et al.,“The biogenesis and functions of microvesicles,” Traffic, vol. 3:321-30(2002)). Fusion of the MVB with the cell membrane results in the releaseof these microvesicles from the cell, and their delivery into the blood,urine or other bodily fluids.

Another category of cell-derived vesicles are known as “sheddingmicrovesicles” (Cocucci et al., “Shedding microvesicles: artefacts nomore,” Trends Cell Biol., vol. 19:43-51 (2009)). These sheddingmicrovesicles, formed by directly budding off of the cell's plasmamembrane, are often heterogeneous in size, and like microvesicles, alsocontain a sample of the parent cell's cytoplasm. Microvesicles andshedding microvesicles co-isolate using ultracentrifugation andultrafiltration isolation techniques and will, therefore, becollectively referred to here as microvesicles.

Vectors based on viruses have shown to be efficient means for genedelivery both to cultured cells and in animal models of human disease.However, limitations to viral vector-mediated gene transfer still exist,including off-target gene delivery/toxicity, inefficient gene deliveryto some cell types, excessive vector uptake by non-target organs, andpre-existing humoral and cell-mediated immunity to the virion capsid.For example, several barriers to efficient gene delivery to the centralnervous system (CNS) exist, including, for example, preexisting immunityto the virus and/or transgene products, the lack of specific receptorson target cell; inefficient or impeded cellular entry/intracellulartrafficking to the nucleus; and the tight junctions of the blood-brainbarrier.

The composition and methods provided herein are designed to overcomethese limitations. The compositions and methods include a viral vector,such as an adenovirus-based vector (replication defective and competentvectors), a lentivirus-based vector, a herpes virus based vector(replication defective and competent vectors), and adeno-associatedvirus based vectors.

The studies provided herein demonstrate that microvesicles isolated fromcell culture media, e.g., cell culture media of adeno-associated virus(AAV) producer cells contain AAV virions. These results were confirmedby electron microscopy. These studies have also demonstrated that thesemicrovesicle fractions containing AAV virons are capable of genetransfer to mammalian cells.

The characteristics of the adeno-associated virus (AAV) allow for itsuse in the methods and compositions provided herein. AAV is a tinynon-enveloped virus having a 25 nm capsid. No disease is known or hasbeen shown to be associated with the wild type virus. AAV has asingle-stranded DNA (ssDNA) genome. The insert capacity of the AAV virusis approximately 4.6 kb. AAV has been shown to exhibit long-termepisomal transgene expression, and AAV has demonstrated excellenttransgene expression in the brain, particularly in neurons. In addition,AAV can exhibit retrograde transport, i.e., from axons to cell body.There are numerous alternative AAV variants (over 100 have been cloned),and AAV variants have been identified based on desirablecharacteristics. For example, AAV9 has been shown to efficiently crossthe blood-brain barrier. Moreover, the AAV capsid can be geneticallyengineered to increase transduction efficient and selectivity, e.g.,biotinylated AAV vectors, directed molecular evolution,self-complementary AAV genomes and so on.

AAV gene therapy is currently being used in treatment of several centralnervous system disorders (e.g., Parkinson's disease, Alzheimer's diseaseand glioma). For example, in one study, six Parkinson's patientsreceived injection of AAV vector containing aromatic L-amino aciddecarboxylase (AADC) gene, and transfer of AADC into the putamen. 96weeks of AADC expression was observed (increased 56%), and motorfunction improved 46%. In another study of children with Leber'scongenital amaurosis (leads to blindness), all children gainedambulatory vision, and one child gained light sensitivity equal to thatof normal individuals.

Vectors based on adeno-associated virus (AAV) have shown remarkableefficiency for gene delivery both to cultured cells and in animal modelsof human disease. However, limitations to AAV vector-mediated genetransfer still exist, including off-target gene delivery/toxicity,inefficient gene delivery to some cell types, excessive vector uptake bynon-target organs, such as liver uptake of AAV after intravenousinjection, and pre-existing humoral and cell-mediated immunity to thevirion capsid, which can abrogate gene delivery and expression in targettissues.

The methods provided herein address the limitations of using AAV vectorsfor gene transfer through the use of microvesicles. The characteristicsof microvesicles are well suited for the compositions and methodsprovided herein. A microvesicle is small, nanometers in size, and cancontain DNA, mRNA and microRNA (miRNA). Microvesicles contain a lipidmembrane and host proteins that are recognized as self by the immunesystem. Microvesicles can encapsulate or otherwise package a widevariety of molecules, including nucleic acids and proteins. Themicrovesicles used in the compositions and methods provided herein areuseful for shielding the viral capsid from pre-existing neutralizingantibodies.

The microvesicles and associated viral vector are produced and purifiedusing methods that differ from the standard procedure for the productionof viral vectors such as AAV. In the standard procedure for AAVproduction, the AAV vectors are produced by triple transfection of 293Tcells with plasmids encoding for structural, nonstructural, and helpervirus genes required for replication and virus production, and then, thevirus is harvested and purified from cell lysates and the media isdiscarded. In the methods and studies described herein, the media fromproducer cells (i.e., cells that produce and shed microvesicles) isharvested, rather than the cells. The microvesicles in the media arepelleted, the pellet is resuspended and then loaded onto a densitygradient. The fractions are collected and analyzed.

In the studies provided herein, the natural ability of cells to secretemicrovesicles was exploited. Following transfection of 293T cells withan AAV2 vector construct and capsid expression cassettes in a standardvector production paradigm, it was discovered that a substantialfraction of AAV virons released from the cells were withinmicrovesicles, e.g., AAV-microvesicles. Intact AAV capsids withinindividual microvesicles were observed by transmission electronmicroscopy.

The studies presented herein also demonstrate that viral vectors such asAAV vectors can be packaged inside or otherwise associated microvesicles(see e.g., FIGS. 4A-4E). In mice, intravenous injection of microvesiclesassociated with AAV2 encoding a luminescent reporter (fireflyluciferase) gave higher whole-body luminescence, as well as uniquetissue selectivity compared to the standard-purified AAV2 vector (seee.g., FIGS. 3A, 3B). Microvesicles represent a unique gene deliveryentity that improves virus vector-based gene therapy.

The studies described herein demonstrate that under normal AAVproduction conditions a detectable amount of AAV virions are associatedwith microvesicles which bud off the surface of the cells into themedium. These microvesicle-associated AAV vectors, termed AAV MAVs, werecapable of gene transfer in cultured cells and vastly surpassed standardAAV vectors of the same serotype both in level and distribution of geneexpression in mice in vivo as assessed by bioluminescence imagingfollowing i.v. injection. The global gene expression observed forAAV2-Fluc microvesicles (FIGS. 3A, 3B) was striking as AAV2 has beenshown to mediate relatively low expression levels restricted mainly tothe liver even at high doses. In contrast, for AAV2-Fluc microvesicles,strong expression was seen in the liver with signal over the entireanimal as early as two weeks and at a low dose (4.4×10⁹ gc/animal).

The purified populations, compositions and methods provided hereinutilize a novel pathway by which viral vectors, including, for example,AAV vectors, are exported from producer cells via an association withmicrovesicles. Isolation and characterization of these packaged orotherwise associated viral vectors (MAV) showed that they displayed invivo gene delivery properties superior to standard viral vectorspurified by standard techniques. The use of packaged or otherwiseassociated viral vectors (MAV) improves the use of this vector platformboth as a tool of molecular biology as well as a gene therapy vector.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the humanheavy chain immunoglobulin molecules and the human light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, 0-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,α-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, the lefthanddirection is the amino terminal direction and the righthand direction isthe carboxy-terminal direction, in accordance with standard usage andconvention.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the nucleic acid sequences arecontemplated as being encompassed by the present invention, providingthat the variations in the nucleic acid sequence maintain at least 75%,more preferably at least 80%, 90%, 95%, and most preferably 99%.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

The term “promoter sequence” as used herein shall mean a polynucleotidesequence comprising a region of a gene at which initiation and rate oftranscription are controlled. A promoter sequence comprises an RNApolymerase binding site as well as binding sites for other positive andnegative regulatory elements. Positive regulatory elements promote theexpression of the gene under control of the promoter sequence. Negativeregulatory elements repress the express of the gene under control of thepromoter sequence. Promoter sequences used herein are found eitherupstream or internal to the gene being regulated. Specifically, the term“first promoter sequence” versus “second promoter sequence” refers tothe relative position of the promoter sequence within the expressionvector. The first promoter sequence is upstream of the second promotersequence.

The term “endogenous gene” as used herein shall mean a gene encompassedwithin the genomic sequence of a cell. The term “exogenous gene” as usedherein shall mean a gene not encompassed within the genomic sequence ofa cell. Exogenous genes are introduced into cells by the instantmethods. The term “transgene” as used herein shall mean a gene that hasbeen transferred from one organism to another.

The term “transfection” as used herein shall mean the transportationacross the cell membrane or insertion of one or more DNA compositionsinto a cell. “Stable transfection” as used herein shall mean thegeneration, under selective pressure, of isolated protein-expressingcell lines. “Semi-stable transfection” as used herein shall mean thegeneration, under selective pressure, of a mixture of protein-expressingcell lines. “Transient transfection” as used herein shall mean thegeneration, without selective pressure, of protein-expressing celllines. Stable and semi-stable transfections may lead to incorporation oftransfected sequences into the genome due to selective pressure.Transient transfections do not lead to genomic incorporation oftransfected sequences and typically retain these sequences for a shorterperiod of time. The term “transfection-resistant” as used herein shallmean transfected with low efficiency or success using known methods.

The term “reporter gene” as used herein shall mean a polynucleotidesequence encoding for a polypeptide that creates a physical change inthose cells which incorporate the expression vector, and, thus, the geneof interest. Physical changes are often color changes or fluorescence.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

As used herein, a “bodily fluid” refers to a sample of fluid isolatedfrom anywhere in the body of the subject, preferably a peripherallocation, including but not limited to, for example, blood, plasma,serum, urine, sputum, spinal fluid, pleural fluid, nipple aspirates,lymph fluid, fluid of the respiratory, intestinal, and genitourinarytracts, tear fluid, saliva, breast milk, fluid from the lymphaticsystem, semen, cerebrospinal fluid, intra-organ system fluid, asciticfluid, tumor cyst fluid, amniotic fluid and combinations thereof.

The term “subject” is intended to include all animals shown to orexpected to have microvesicles. In particular embodiments, the subjectis a mammal, a human or nonhuman primate, a dog, a cat, a horse, a cow,other farm animals, or a rodent (e.g. mice, rats, guinea pig, etc.). Theterm “subject” and “individual” are used interchangeably herein.

Methods of isolating microvesicles from a biological sample are known inthe art. For example, a method of differential centrifugation isdescribed in a paper by Raposo et al. (Raposo et al., 1996), and similarmethods are detailed in the Examples section herein. Methods of anionexchange and/or gel permeation chromatography are described in U.S. Pat.Nos. 6,899,863 and 6,812,023. Methods of sucrose density gradients ororganelle electrophoresis are described in U.S. Pat. No. 7,198,923. Amethod of magnetic activated cell sorting (MACS) is described in (Taylorand Gercel-Taylor, 2008). A method of nanomembrane ultrafiltrationconcentrator is described in (Cheruvanky et al., 2007).

Preferably, microvesicles can be identified and isolated from bodilyfluid of a subject by a newly developed microchip technology that uses aunique microfluidic platform to efficiently and selectively separatetumor derived microvesicles. This technology, as described in a paper byNagrath et al. (Nagrath et al., 2007), can be adapted to identify andseparate microvesicles using similar principles of capture andseparation as taught in the paper. Each of the foregoing references isincorporated by reference herein for its teaching of these methods.

In one embodiment, the microvesicles isolated from a bodily fluid areenriched for those originating from a specific cell type, for example,lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis,skin, colorectal, breast, prostate, brain, esophagus, liver, placenta,fetus cells. Because the microvesicles often carry surface moleculessuch as antigens from their donor cells, surface molecules may be usedto identify, isolate and/or enrich for microvesicles from a specificdonor cell type (Al-Nedawi et al., 2008; Taylor and Gercel-Taylor,2008). In this way, microvesicles originating from distinct cellpopulations can be analyzed for their nucleic acid content. For example,tumor (malignant and non-malignant) microvesicles carry tumor-associatedsurface antigens and may be detected, isolated and/or enriched via thesespecific tumor-associated surface antigens. In one example, the surfaceantigen is epithelial-cell-adhesion-molecule (EpCAM), which is specificto microvesicles from carcinomas of lung, colorectal, breast, prostate,head and neck, and hepatic origin, but not of hematological cell origin(Balzar et al., 1999; Went et al., 2004). In another example, thesurface antigen is CD24, which is a glycoprotein specific to urinemicrovesicles (Keller et al., 2007). In yet another example, the surfaceantigen is selected from a group of molecules CD70, carcinoembryonicantigen (CEA), EGFR, EGFRvIII and other variants, Fas ligand, TRAIL,transferrin receptor, p38.5, p97 and HSP72. Additionally, tumor specificmicrovesicles may be characterized by the lack of surface markers, suchas CD80 and CD86.

The isolation of microvesicles from specific cell types can beaccomplished, for example, by using antibodies, aptamers, aptameranalogs or molecularly imprinted polymers specific for a desired surfaceantigen. In one embodiment, the surface antigen is specific for a cancertype. In another embodiment, the surface antigen is specific for a celltype which is not necessarily cancerous. One example of a method ofmicro vesicle separation based on cell surface antigen is provided inU.S. Pat. No. 7,198,923. As described in, e.g., U.S. Pat. Nos. 5,840,867and 5,582,981, WO/2003/050290 and a publication by Johnson et al.(Johnson et al., 2008), aptamers and their analogs specifically bindsurface molecules and can be used as a separation tool for retrievingcell type-specific microvesicles. Molecularly imprinted polymers alsospecifically recognize surface molecules as described in, e.g., U.S.Pat. Nos. 6,525,154, 7,332,553 and 7,384,589 and a publication by Bossiet al. (Bossi et al., 2007) and are a tool for retrieving and isolatingcell type-specific microvesicles. Each of the foregoing reference isincorporated herein for its teaching of these methods.

It may be beneficial or otherwise desirable to extract the nucleic acidfrom the microvesicles for analysis. Nucleic acid molecules can beisolated from a microvesicle using any number of procedures, which arewell-known in the art, the particular isolation procedure chosen beingappropriate for the particular biological sample. In some instances,with some techniques, it may also be possible to analyze the nucleicacid without extraction from the microvesicle.

In one embodiment, the extracted nucleic acids, including DNA and/orRNA, are analyzed directly without an amplification step. Directanalysis may be performed with different methods including, but notlimited to, the nanostring technology. NanoString technology enablesidentification and quantification of individual target molecules in abiological sample by attaching a color coded fluorescent reporter toeach target molecule. This approach is similar to the concept ofmeasuring inventory by scanning barcodes.

Reporters can be made with hundreds or even thousands of different codesallowing for highly multiplexed analysis. The technology is described ina publication by Geiss et al. (Geiss et al., 2008) and is incorporatedherein by reference for this teaching.

In another embodiment, it may be beneficial or otherwise desirable toamplify the nucleic acid of the microvesicle prior to analyzing it.Methods of nucleic acid amplification are commonly used and generallyknown in the art, many examples of which are described herein. Ifdesired, the amplification can be performed such that it isquantitative. Quantitative amplification will allow quantitativedetermination of relative amounts of the various nucleic acids, togenerate a profile as described below.

In one embodiment, the extracted nucleic acid is RNA. RNAs are thenpreferably reverse-transcribed into complementary DNAs before furtheramplification. Such reverse transcription may be performed alone or incombination with an amplification step. One example of a methodcombining reverse transcription and amplification steps is reversetranscription polymerase chain reaction (RT-PCR), which may be furthermodified to be quantitative, e.g., quantitative RT-PCR as described inU.S. Pat. No. 5,639,606, which is incorporated herein by reference forthis teaching.

Nucleic acid amplification methods include, without limitation,polymerase chain reaction (PCR) (U.S. Pat. No. 5,219,727) and itsvariants such as in situ polymerase chain reaction (U.S. Pat. No.5,538,871), quantitative polymerase chain reaction (U.S. Pat. No.5,219,727), nested polymerase chain reaction (U.S. Pat. No. 5,556,773),self sustained sequence replication and its variants (Guatelli et al.,1990), transcriptional amplification system and its variants (Kwoh etal., 1989), Qb Replicase and its variants (Miele et al., 1983), cold-PCR(Li et al., 2008) or any other nucleic acid amplification methods,followed by the detection of the amplified molecules using techniqueswell known to those of skill in the art. Especially useful are thosedetection schemes designed for the detection of nucleic acid moleculesif such molecules are present in very low numbers. The foregoingreferences are incorporated herein for their teachings of these methods.

Diseases or other medical conditions for which the inventions describedherein are applicable include, but are not limited to, nephropathy,diabetes insipidus, diabetes type I, diabetes II, renal diseaseglomerulonephritis, bacterial or viral glomerulonephritides, IgAnephropathy, Henoch-Schonlein Purpura, membranoproliferativeglomerulonephritis, membranous nephropathy, Sjogren's syndrome,nephrotic syndrome minimal change disease, focal glomerulosclerosis andrelated disorders, acute renal failure, acute tubulointerstitialnephritis, pyelonephritis, GU tract inflammatory disease, Pre-clampsia,renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis,genetic renal disease, medullary cystic, medullar sponge, polycystickidney disease, autosomal dominant polycystic kidney disease, autosomalrecessive polycystic kidney disease, tuberous sclerosis, vonHippel-Lindau disease, familial thin-glomerular basement membranedisease, collagen III glomerulopathy, fibronectin glomerulopathy,Alport's syndrome, Fabry's disease, Nail-Patella Syndrome, congenitalurologic anomalies, monoclonal gammopathies, multiple myeloma,amyloidosis and related disorders, febrile illness, familialMediterranean fever, HIV infection-AIDS, inflammatory disease, systemicvasculitides, polyarteritis nodosa, Wegener's granulomatosis,polyarteritis, necrotizing and crecentic glomerulonephritis,polymyositis-dermatomyositis, pancreatitis, rheumatoid arthritis,systemic lupus erythematosus, gout, blood disorders, sickle celldisease, thrombotic thrombocytopenia purpura, Fanconi's syndrome,transplantation, acute kidney injury, irritable bowel syndrome,hemolytic-uremic syndrome, acute corticol necrosis, renalthromboembolism, trauma and surgery, extensive injury, burns, abdominaland vascular surgery, induction of anesthesia, side effect of use ofdrugs or drug abuse, circulatory disease myocardial infarction, cardiacfailure, peripheral vascular disease, hypertension, coronary heartdisease, non-atherosclerotic cardiovascular disease, atheroscleroticcardiovascular disease, skin disease, psoriasis, systemic sclerosis,respiratory disease, COPD, obstructive sleep apnoea, hypoia at highaltitude or erdocrine disease, acromegaly, diabetes mellitus, ordiabetes insipidus.

The cancer diagnosed, monitored or otherwise profiled, can be any kindof cancer. This includes, without limitation, epithelial cell cancerssuch as lung, ovarian, cervical, endometrial, breast, brain, colon andprostate cancers. Also included are gastrointestinal cancer, head andneck cancer, non-small cell lung cancer, cancer of the nervous system,kidney cancer, retina cancer, skin cancer, liver cancer, pancreaticcancer, genital-urinary cancer and bladder cancer, melanoma, andleukemia. In addition, the methods and compositions of the presentinvention are equally applicable to detection, diagnosis and prognosisof non-malignant tumors in an individual (e.g. neurofibromas,meningiomas and schwannomas).

In some embodiments, the cancer is brain cancer. Types of brain tumorsand cancer are well known in the art. Glioma is a general name fortumors that arise from the glial (supportive) tissue of the brain.Gliomas are the most common primary brain tumors. Astrocytomas,ependymomas, oligodendrogliomas, and tumors with mixtures of two or morecell types, called mixed gliomas, are the most common gliomas. Thefollowing are other common types of brain tumors: Acoustic Neuroma(Neurilemmoma, Schwannoma, Neurinoma), Adenoma, Astracytoma, Low-GradeAstrocytoma, giant cell astrocytomas, Mid- and High-Grade Astrocytoma,Recurrent tumors, Brain Stem Glioma, Chordoma, Choroid Plexus Papilloma,CNS Lymphoma (Primary Malignant Lymphoma), Cysts, Dermoid cysts,Epidermoid cysts, Craniopharyngioma, Ependymoma Anaplastic ependymoma,Gangliocytoma (Ganglioneuroma), Ganglioglioma, Glioblastoma Multiforme(GBM), Malignant Astracytoma, Glioma, Hemangioblastoma, Inoperable BrainTumors, Lymphoma, Medulloblastoma (MDL), Meningioma, Metastatic BrainTumors, Mixed Glioma, Neurofibromatosis, Oligodendroglioma. Optic NerveGlioma, Pineal Region Tumors, Pituitary Adenoma, PNET (PrimitiveNeuroectodermal Tumor), Spinal Tumors, Subependymoma, and TuberousSclerosis (Bourneville's Disease).

The “pharmaceutically acceptable carrier” means any pharmaceuticallyacceptable means to mix and/or deliver the targeted delivery compositionto a subject. This includes a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agents from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and is compatible with administration to a subject,for example a human.

Administration to the subject can be either systemic or localized. Thisincludes, without limitation, dispensing, delivering or applying anactive compound (e.g. in a pharmaceutical formulation) to the subject byany suitable route for delivery of the active compound to the desiredlocation in the subject, including delivery by either the parenteral ororal route, intramuscular injection, subcutaneous/intradermal injection,intravenous injection, buccal administration, transdermal delivery andadministration by the rectal, colonic, vaginal, intranasal orrespiratory tract route.

It should be understood that this invention is not limited to theparticular methodologies, protocols and reagents, described herein andas such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Therapeutic Administration and Formulations

It will be appreciated that administration of the purified populationsof microvesicles associated with viral particles (MAV) in accordancewith the invention will be administered with suitable carriers,excipients, and other agents that are incorporated into formulations toprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences (15th ed, Mack Publishing Company, Easton, Pa. (1975)),particularly Chapter 87 by Blaug, Seymour, therein. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-waterand water-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. Any of the foregoing mixtures may be appropriate intreatments and therapies in accordance with the present invention,provided that the active ingredient in the formulation is notinactivated by the formulation and the formulation is physiologicallycompatible and tolerable with the route of administration. See alsoBaldrick P. “Pharmaceutical excipient development: the need forpreclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000),Wang W. “Lyophilization and development of solid proteinpharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N“Lipids, lipophilic drugs, and oral drug delivery-some emergingconcepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendiumof excipients for parenteral formulations” PDA J Pharm Sci Technol.52:238-311 (1998) and the citations therein for additional informationrelated to formulations, excipients and carriers well known topharmaceutical chemists.

The purified populations of microvesicles associated with viralparticles (MAV) are administered to a subject, for example, a subject inneed of gene therapy. Subjects are identified using any of a variety ofclinical and/or laboratory tests such as, physical examination,radiologic examination and blood, urine and stool analysis to evaluateimmune status.

Administration of the purified populations of microvesicles associatedwith viral particles (MAV) to a patient is considered successful if anyof a variety of laboratory or clinical results is achieved. For example,administration of the purified populations of microvesicles associatedwith viral particles (MAV) to a patient is considered successful one ormore of the symptoms associated with the disorder afflicting the patientis alleviated, reduced, inhibited or does not progress to a further,i.e., worse, state. Administration of the purified populations ofmicrovesicles associated with viral particles (MAV) to a patientsuffering from a disorder such is considered successful if the disorderenters remission or does not progress to a further, i.e., worse, state.

In some embodiments, the purified populations of microvesiclesassociated with viral particles (MAV) are administered in combinationwith a second agent, including for example, any of a variety of knownanti-inflammatory and/or immunosuppressive compounds. In someembodiments, the purified populations of microvesicles associated withviral particles (MAV) are used in conjunction with a surgical method oftreating or otherwise alleviating the disorder.

The purified populations of microvesicles associated with viralparticles (MAV) are administered to a subject in an amount sufficient tohave a desired modulation effect in the subject. In some embodiments,administration of the purified populations of microvesicles associatedwith viral particles (MAV) will abrogate or inhibit or otherwiseinterfere with at least one biological property and/or biologicalactivity of a target.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1: Materials & Methods

Cell culture: 293T human cells, U87 human glioma cells (both fromAmerican Type Culture Collection, Manassas, Va.) and Gli36 human gliomacells (kindly provided by Dr. Anthony Capanogni, University ofCalifornia at Los Angeles, Los Angeles, Calif.), were maintained inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetalbovine serum (Sigma, St. Louis, Mo.), 100 U/ml penicillin, and 0.1 mg/mlstreptomycin (Sigma), referred to as complete DMEM. All cells were grownat 37° C. in a 5% CO₂ humidified atmosphere.

Vector production and microvesicle isolation: AAV vectors were producedby transfecting 15 cm plates of 293T cells using the calcium phosphatemethod with the following plasmids: 12 μg of an ITR-flanked AAVtransgene expression vector (green fluorescent protein, GFP or fireflyluciferase, Fluc), 25 μg of an adenovirus helper plasmid Fd6 (MolecularTherapy (2005) 11, 843-848), and 12 μg of the AAV2 rep/cap expressionvector, pH22 (Virology. February 2003, p. 2768-2774, Vol. 77, No. 4), orthe AAV2rep/AAV1cap expression vector, pXR1 (from the UNC Gene TherapyCenter). Sixteen hours post-transfection media was exchanged with freshmedia supplemented with 2% microvesicle-depleted fetal bovine sera, FBS.Forty-eight to seventy-two hours post transfection media was harvestedand microvesicles were purified as described below in “gradientcentrifugation.” Standard AAV was generated as previously described(Maguire C A et al., J Neurooncol. 2010 February; 96(3):337-47). Thevector titer (in genome copies/ml [gc/ml]) was performed using aquantitative TaqMan PCR assay. First, AAV genomic DNA was isolated bytreating a 2 μl microvesicle fraction in a 50 μg reaction with 2 unitsof DNaseI for 2 h at 37° C. to remove any potential extravesicular,unencapsidated AAV genomes or plasmid DNA. Next, DNase was inactivatedfor 25 min at 75° C. Then a PCR reaction was prepared using TaqMan® FastUniversal PCR Master Mix (Applied Biosystems, Foster City, Calif.), aTaqMan probe (5′-6FAM-TGCCAGCCATCTGTTGTTTGCC-MGB (SEQ ID NO: 1) AppliedBiosystems) and primer set (forward primer, 5′-CCTCGACTGTGCCTTCTAG-3′(SEQ ID NO: 2); reverse primer, 5′-TGCGATGCAATTTCCTCAT-3′ (SEQ ID NO:3)) which specifically anneals to the Poly A signal sequence region inthe transgene cassette. A standard curve was prepared using serialdilutions of an AAV plasmid of a known molar concentration. Thequantitative PCR was performed in an Applied Biosystems 7500Thermalcycler using the following conditions: 1 cycle, 94° C. 30 s; 40cycles 94° C. 3 s, 60° C. 30 s.

Gradient centrifugation: Microvesicles were purified using a 6-18%iodixanol gradient as previously described (Cantin et al.) with thefollowing modifications. The media harvested from AAV producer cells(293T) was centrifuged for 10 min. at 600×g. Media was then transferredto a fresh tube and centrifuged at 20,000×g for 25 min. The resultingmicrovesicle pellet was resuspended in 1 ml of phosphate buffered salineand treated with 250 U of Benzonase nuclease (Sigma) for 1 h at 37° C. A6-18% iodixanol step gradient was prepared in 38.5 ml open-topthin-walled polyallomer tubes (Beckman, Palo Alto, Calif.) usingOptiprep (Sigma-Aldrich, St. Lois, Mo.). After overlaying the gradientwith one ml of the resuspended microvesicle pellet, the tubes werecentrifuged at 32,000 rpm in a SW32 Ti rotor (Beckman-Coulter) for 129min with the brake off in an Optima L-90K ultracentrifuge(Beckman-Coulter). Following the spin, the top 2 ml of the gradient werediscarded and then 1 ml fractions from the top to the bottom of thegradient were collected.

Immunoblotting: After titration, a volume containing 10⁸ gc of AAV ofthe iodixanol gradient fraction was mixed with and added to SDS loadingbuffer. Samples were heated at 95° C. for 5 minutes. SDS-PAGE wasperformed using NuPAGE Novex 10% Bis-Tris acrylamide gels and MESrunning buffer. The gel was electrophoresed at 130V for 90 min.Following transfer of proteins to a nitrocellulose membrane, themembrane was blocked for 1 h in 10% milk in TBS/0.1% Tween-20. For AAVcapsid protein immunoblotting membranes were probed with a 1:100dilution of anti-adeno-associated virus VP1, VP2, VP3 antibody (AmericanResearch Products, Belmont, Mass.). A 1:5,000 dilution of anti-rabbitHRP conjugated secondary antibody (GE Healthcare) was used. Specificbinding was detected using Pierce SuperSignal West Pico chemiluminescentsubstrate (Thermoscientific, Rockford, Ill.) and exposure of HyBlot CLautoradiography film (Denville Scientific, South Plainfield N.J.) to themembrane.

Vector transduction assays: 10⁴ cells (cell type indicated in figure)were plated the day before transduction in 96 well plates. Cell lysate(standard) purified AAV2 encoding firefly luciferase (Fluc), termedAAV2-Fluc, or microvesicle-associated AAV2-Fluc were mixed with media(10⁴ gc/cell) in a total volume of 500 μl added to cells. One hourlater, media was removed and replaced with fresh media. After 48-72 hincubation at 37° C., cells were rinsed in PBS, lysed using ReporterLysis Buffer (Promega, Madison, Wis.). A luciferase assay was performedon 20 μl of lysate using a luminometer equipped with an injector thatadded 100 μl of luciferase substrate buffer/well. Luciferase activitieswere normalized to protein content of the samples by performing aBradford assay (BioRad, Hercules, Calif.).

GAPDH reverse transcriptase PCR: To detect mRNA inside microvesicles,10⁸ g.c. of purified AAV2-Fluc associated microvesicles, were used toisolate RNA using Trizol LS reagent (Invitrogen). For a positivecontrol, RNA was isolated from HeLa cells. Next a two-step reversetranscriptase PCR was performed using Sensiscript reverse transcriptase(Qiagen) and HotStarTaq DNA polymerase (Qiagen) was performed to amplifyGAPDH transcripts from microvesicles RNA or from HeLa RNA. cDNAsynthesis: 50 ng of RNA was used for template performed with Sensiscriptreverse transcriptase (Qiagen). PCR amplification of GAPDH cDNA: 1 cycle95° C. for 3 min; 40 cycles, 95° C. for 30 sec, 60° C. for 30 s, 70° C.for 30 s; 1 cycle 70° C. for 7 min. PCR products were analyzed on a 2%ethidium bromide-stained agarose gel.

RNA analysis for small RNA species: Extracted RNA was examined for smallRNA species using a small RNA Bioanalyzer chip.

Nanoparticle tracking analysis (NTA): The size and concentration ofnanoparticles in the microvesicle preparations was determined using aLM10-HS nanoparticle analyzer (Nanosight, Amesbury, UK) operated by NTAsoftware. Samples were diluted in phosphate buffered saline and theparticle size, particle concentration, and particle size distributionmeasured.

Example 2. Isolation, Purification and Characterization of MicrovesiclesContaining Viral Vectors

293T cells were transfected with AAV and helper plasmids to generate AAVvectors encoding green fluorescent protein (GFP). Forty-eight hourslater, cells were prepared for transmission electron microscopy. Usingplastic, embedded sectioned samples, microvesicles were observed on theoutside of AAV-producer cells. Microvesicles were detected near the 293Tproducer cell surface. AAV particles were observed inside themicrovesicles. Capsid-like structures were measured using software anddetermined to be 20-25 nm, which is the expected size of the AAV2capsid.

In the methods used herein, the media was harvested from 293T cellsproducing AAV vectors and centrifuged at 300×g for 10 minutes to removecells and other debris. The microvesicles and associated AAV werepelleted by centrifuging 20,000×g for 25 minutes, and the pellet wasthen resuspended in phosphate buffered saline and treated for 1 h at 37°C. with Benzonase to remove unprotected AAV DNA from transfection. Thesample was then loaded onto a 6-18% iodixanol step gradient (in 1.2%step increments) and centrifuged in a SW32Ti swinging bucket rotator(Beckman Coulter) for 2 h 9 min at 32,000 rpm (no brake). The gradientwas fractionated from top.

Standard purified AAV, i.e., cell lysates AAV, does not pelletefficiently using the centrifugation speed that is used for microvesiclepelleting (FIG. 1). Iodixanol-purified AAV isolated from cell lysates,which was presumably not associated with microvesicles, was centrifugedin DMEM 10% FBS containing media for 20,000×g for 25 minutes. Pelletingefficiency was determined by qPCR titration of AAV genomes in the mediapre and post-centrifugation.

The AAV-microvesicle pellet was found to exhibit a differentsedimentation velocity in iodixanol gradient as compared to cell lysatepurified vector. Media from AAV producer cells (AAV vector encodesfirefly luciferase (AAV-fluc)) was centrifuged for 25 min at 20,000×gand the media and microvesicle pellet were divided into separate samplesand each loaded onto a separate 6-18% iodixanol step gradient. Forcomparison, cell lysate purified AAV-fluc was mixed with purifiednon-transfected 293T microvesicles and centrifuge on the gradient asdescribed above. After centrifugation, each sample's gradient wasdivided into 1 ml fractions. An aliquot of each fraction was then addedto separate wells containing 293T cells, and 48 hrs later cells werelysed and a luciferase assay performed to determine the fractionscontaining peak transgene (flue) expression, Fraction 36.

Microvesicles from Fraction 36 were analyzed for size distribution bynanoparticle tracking analysis using a Nanosight particle analyzer. Alarge vesicle fraction was observed in the AAV associated-microvesiclesamples but not the cell lysate purified AAV+microvesicle mix. Theconcentration of microvesicles was much higher in media from AAVproducer cells as compared to nontransfected 293T.

The AAV associated-microvesicle pellet was shown to contain small RNAspecies in contrast to the cell lysates purified AAV and cell lysatepurified AAV⁺ microvesicle mix. Microvesicles from fraction 36 wereanalyzed for the presence of small RNA species characteristic ofmicrovesicles using a Bioanalyzer.

The AAV associated-microvesicles were shown to contain mRNA.Microvesicle Fraction 36 was analyzed for Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) mRNA using reverse transcriptase PCR. RNA wasextracted from a sample volume containing 1e8 genome copies of AAV asdetermined by quantitative real time PCR. A specific band of around 200bp was seen in samples containing microvesicles.

It was observed that the AAV associated-microvesicles contained AAVcapsid proteins. A volume containing 1e8 genome copies of AAV from themicrovesicle sample from Fraction 36 was loaded onto an SDS-PAGE gel. Animmunoblot was performed using an anti-AAV capsid antibody to detect thethree virus proteins, VP1, VP2, and VP3.

The AAV associated-microvesicles were shown to contain AAV genomes usinganalysis by quantitative PCR to detect the presence of AAV genomes.

The AAV associated-microvesicles efficiently transduced cells in culture(FIG. 2). Cell-lysate purified AAV or AAV associated-microvesicles fromFraction 36 were used at equal doses (10⁴ gc/cell) to transduce variouscells in culture. The vectors encoded firefly luciferase (fluc) as ameans of enabling measurement of transgene delivery and expression 48hours post treatment.

To increase the yield of AAV associated-microvesicles, a DNA plasmidencoding vesicular stomatitis virus glycoprotein G (VSV-G) wasco-transfected during AAV production in 293T cells. In three independentAAV associated-microvesicle preparations, the quantitative PCR genomecopy titers in the presence and absence of the VSV-G plasmid duringtransfection were compared. A 5-65 fold enhancement in titer wasobserved when using the VSV-G plasmid.

Example 3: In Vivo Gene Expression in Purified AAVAssociated-Microvesicles

In the studies described herein, the media from AAV producer cells washarvested 72 h post-transfection and spun at 300×g 10 min to removecells and other debris. The AAV associated-microvesicles were pelletedby spinning at 20,000×g for 25 min. The pellet was then resuspended in1×PBS (500 μl), and 50 μl 10× TURBO DNase Buffer and 10 μl Turbo DNasewere added. The mixture was incubated for 30 min at 37° C. Then, 110 μlof resuspended DNase Inactivation reagent was added and mixedthoroughly. The mixture was then mixed occasionally at room temperaturefor 5 min. The mixture was then centrifuged at 2,000×g for 1.5 min, andthe supernatant was transferred to a fresh tube. The supernatant waskept on ice, titered and injected into mice.

2×10⁹ genome copies of standard cell lysate purified AAV2-fluc orAAV2-fluc AAV associated-microvesicles were injected via the tail veinof nude mice. Two weeks post injection, d-luciferin was injectedintraperitoneally, and fluc expression was detected with bioluminescenceimager (FIGS. 3A, 3B).

Example 4: Immunogold Labeling and Detection of AAV AssociatedMicrovesicles

Cryosections of microvesicle associated vectors that contained either anAAV1 vector or an AAV2 vector (i.e., both AAV1 and AAV2 serotypes weretested) were stained with commercially available anti-AAV1 and anti-AAV2antibodies followed by a secondary antibody labeled with 10 nm gold. Inthis study, the anti-AAV2 antibody served as a negative control forAAV1, while the anti-AAV1 antibody served as a negative control forAAV2. No immunogold labeling was observed for negative controls. Thesections were then imaged by transmission electron microscopy.

The results of this study are shown in FIGS. 4A-4E. In these figures,the arrows are indicating the location of AAV (AAV1 or AAV2) that is“free,” i.e., outside of a microvesicle, the triangles are indicatingthe location of AAV (AAV1 or AAV2) inside a microvesicle, and thediamond-headed arrows are indicating the location of AAV (AAV1 or AAV2)that is bound to surface of a microvesicle. As shown in FIGS. 4A-4E, AAVcapsids were detected inside microvesicles for both AAV1 and AAV2serotypes, and a lot of free AAV capsids were also detected.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method of producing a purified population ofmicrovesicles associated with one or more adeno-associated virus (AAV)vector particles comprising a transgene cassette, comprising the steps:a) engineering producer cells to produce the AAV vector particles; andb) purifying microvesicles shed from the engineered producer cells; tothereby produce the population of microvesicles.
 2. The method of claim1, further comprising the step of increasing production of microvesiclesfrom the producer cells.
 3. The method of claim 2, wherein increasingproduction of microvesicles from the producer cells is by exposing theproducer cells to a stimulus following engineering step a).
 4. Themethod of claim 3, wherein the stimulus is a chemical stressor or anenvironmental stressor.
 5. The method of claim 1, wherein the producercells have been genetically engineered to increase their production ofmicrovesicles.
 6. The method of claim 5, wherein the producer cells havebeen genetically engineered to express vesicular stomatitis virusglycoprotein G (VSV-G).
 7. The method of claim 1, wherein engineeringstep a) is by transfection of the producer cells with plasmids encodingAAV structural, AAV nonstructural and helper virus genes.
 8. The methodof claim 1, wherein the producer cells express a ligand or receptor thatis incorporated into the outer surface of the microvesicles.
 9. Themethod of claim 1, wherein the producer cells further comprise atherapeutic molecule that is incorporated into the microvesicles withthe AAV vector particle.
 10. The method of claim 9, wherein thetherapeutic molecule is a protein or RNA.
 11. The method of claim 10,wherein the RNA is RNAi, shRNA, siRNA, miRNA, or a combination thereof.